Identification and characterization of a novel cytoskeleton-associated pp60src substrate.

Abstract

Transformation of cells by the src oncogene results in elevated tyrosine phosphorylation of two related proteins, p80 and p85 (p80/85). Immunostaining with specific monoclonal antibodies revealed a striking change of subcellular localization of p80/85 in src-transformed cells. p80/85 colocalizes with F-actin in peripheral extensions of normal cells and rosettes (podosomes) of src-transformed cells. Sequence analysis of cDNA clones encoding p80/85 revealed an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids, a carboxyl-terminal SH3 domain, and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The multidomain structure of p80/85 and its colocalization with F-actin in normal and src-transformed cells suggest that these proteins may associate with components of the cytoskeleton and contribute to organization of cell structure.Transformation of cells by tyrosine kinase oncogenes leads to alterations of cell shape, cellular metabolism, growth control, and gene expression (16,31,54). A substantial body of evidence indicates that many if not all of these changes are a direct result of the tyrosine kinase activity of the oncogene product (reviewed in references 31, 54, and 66). Rous sarcoma virus (RSV) encodes an enzymatically activated, 60-kDa tyrosine protein kinase, pp60v"src (6,15,44). However, the product of the normal cellular homolog of src, pp60c-sc, is enzymatically down regulated and does not induce significant alterations in cellular growth or changes in cell morphology when overexpressed in rodent or avian cells (30,53,67). Oncogenic activation of c-src and concomitant activation of tyrosine kinase activity can be achieved by mutation of the regulatory site of tyrosine phosphorylation, 40,58,63). Thus, expression of pp6Ov-src or activated forms of the c-src protein pp60527F results in efficient cellular transformation and the increased tyrosine phosphorylation of approximately 15 to 30 cellular proteins (27,33,45,61).Genetic studies have shown that structural perturbation of several different domains of pp6Osrc leads to alterations in the pattern of tyrosine phosphorylation of specific cellular proteins and accompanying changes in morphological phenotypes (reviewed in references 31 and 54). For example, mutation of the site of myristylation (e.g., Gly-2 to Ala) of pp60v-src or pp60527F blocks cellular transformation (9, 32, 61) and the tyrosine phosphorylation of a 120-kDa cellular protein (35,45,61). pp60src contains two regions that share amino acid sequence similarity with other nonreceptor tyrosine protein kinases (55) and regulatory proteins such as phospholipase C--y, Crk, and GTPase-activating protein (GAP) (70,73,76). Structural alterations within these regions alter or abolish the transforming activity of src (18,28,51,59,77,79) Whereas recent experiments have shown that tyrosine phosphorylation of some cellular proteins appears to direct stable protein-protein interactio...

Overexpression of EMS1/cortactin in NIH3T3 fibroblasts causes increased cell motility and invasion in vitro

“…Interestingly, cyclin D1, which also maps to the 11q13 amplicon, has been shown to associate with cyclin-dependent kinases (CDK), CDK4 and CDK6 in a HNSCC cell line (Bates et al, 1994). Thus, we speculate that CDKs associated with cyclin D1 might phosphorylate cortactin at consensus CDK phosphorylation sites Wu et al, 1991). Such an event may be one means by which cyclin D1 and EMS1 genes cooperate in the development and progression of tumors with 11q13 ampli®cation by promoting cell cycle progression and tumor cell migration/invasion, respectively.…”

“…In comparison, the consequences of cortactin overexpression remain speculative. Cortactin has been demonstrated to be an actin-binding protein and one of the major phosphotyrosine proteins present in cells transformed by the src tyrosine kinase oncogene (Kanner et al, 1990;Wu et al, 1991). Localization studies have shown cortactin associated with the cytoskeleton and cell contact sites, suggesting that overexpression of cortactin may in¯uence cell adhesion, migration and/ or invasive potential.…”

Tumorigenesis mediated by MET mutant M1268T is inhibited by dominant-negative Src

“…The majority of the transforming v-Src is located in cytoskeleton and phosphorylates the components of the cytoskeleton (Hamaguchi et al, 1987;Sefton et al, 1981;Pasquale et al, 1986;Gould et al, 1986;Wu et al, 1991;Turner et al, 1990). Normally, c-Src is associated with inner plasma membrane or with endosomal membrane, not cytoskeleton.…”

Human cortactin as putative cancer antigen

“…This region, which apparently includes the epitope recognized by B cells, contains an SH3 domain at the C-terminus and ®ve and-a-half tandemly repeated copies of 37 amino acid residues (out of six and-a-half present in the full-size cortactin protein) that are believed to mediate binding of cortactin to ®lamentous actin in vitro. No evidence of mutations in this region was found in clone MOBr-140 as compared to published cortactin gene sequence (Schuuring et al, 1993(Schuuring et al, , 1998.Human cortactin is a p80/p85 multidomain actin ®lament-binding protein (Schuuring et al, 1993) ®rst identi®ed as a src kinase substrate in chicken ®broblasts (Wu et al, 1991). Cortactin is involved in the signaling pathway of mitogenic receptors and adhesion molecules mediating cytoskeletal reorganization (Schuuring et al, 1993(Schuuring et al, , 1998van Damme et al, 1997;Campbell et al, 1999).…”

“…Human cortactin is a p80/p85 multidomain actin ®lament-binding protein (Schuuring et al, 1993) ®rst identi®ed as a src kinase substrate in chicken ®broblasts (Wu et al, 1991). Cortactin is involved in the signaling pathway of mitogenic receptors and adhesion molecules mediating cytoskeletal reorganization (Schuuring et al, 1993(Schuuring et al, , 1998van Damme et al, 1997;Campbell et al, 1999).…”